Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2008/087258 filed on Dec. 17, 2008 and claims priority from U.S.provisional application Ser. No. 61/014,999 filed on Dec. 19, 2007,which applications are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity and isolated polynucleotides encodingthe polypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

It would be advantageous in the art to improve the ability to convertcellulosic feedstocks.

WO 2005/074647 discloses isolated polypeptides having cellulolyticenhancing activity and polynucleotides thereof from Thielavia terrestrisWO 2005/074656 discloses an isolated polypeptide having cellulolyticenhancing activity and the polynucleotide thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated polypeptide havingcellulolytic enhancing activity and the polynucleotide thereof fromTrichoderma reesei.

The present invention relates to polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 60%identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, or (iii) afull-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 60% identity to the mature polypeptide codingsequence of SEQ ID NO: 1 and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having cellulolytic enhancing activity, selected from thegroup consisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence having at least 60% identity to the mature polypeptide of SEQID NO: 2:

(b) a polynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii):

(c) a polynucleotide comprising a nucleotide sequence having at least60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingcellulolytic enhancing activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. The presentalso relates to such a double-stranded inhibitory RNA (dsRNA) molecule,wherein optionally the dsRNA is a siRNA or a miRNA molecule.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with a cellulolytic enzyme composition in the presence of sucha polypeptide having cellulolytic enhancing activity, wherein thepresence of the polypeptide having cellulolytic enhancing activityincreases the degradation of cellulosic material compared to the absenceof the polypeptide having cellulolytic enhancing activity.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with a cellulolytic enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity, wherein the presenceof the polypeptide having cellulolytic enhancing activity increases thedegradation of cellulosic material compared to the absence of thepolypeptide having cellulolytic enhancing activity; (b) fermenting thesaccharified cellulosic material of step (a) with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with a cellulolytic enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention and the presence of the polypeptide having cellulolyticenhancing activity increases the degradation of the cellulosic materialcompared to the absence of the polypeptide having cellulolytic enhancingactivity.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding a polypeptide having cellulolytic enhancingactivity.

The present invention also relates to methods of producing a polypeptidehaving cellulolytic enhancing activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide having cellulolytic enhancing activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of amino acids 1 to 15 of SEQ ID NO: 2, wherein the gene isforeign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence and the deduced amino acidsequence of a Myceliophthora thermophila CBS 202.75 GH61I polypeptidehaving cellulolytic enhancing activity (SEQ ID NOs: 1 and 2,respectively).

FIG. 2 shows a restriction map of pSMai193.

FIG. 3 shows a restriction map of pSMai189.

DEFINITIONS

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity that enhances thehydrolysis of a cellulosic material by proteins having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars orin the increase of the total of cellobiose and glucose from thehydrolysis of a cellulosic material by cellulase protein under thefollowing conditions: 1-50 mg of total protein/g of cellulose in PCS,wherein total protein is comprised of 80-99.5% w/w cellulase protein/gof cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancingactivity for 1-7 days at 50° C. compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferredaspect, a mixture of CELLUCLAST®1.5 L (Novozymes A/S, Bagsvaerd,Denmark) in the presence of 3% of total protein weight Aspergillusoryzae beta-glucosidase (recombinantly produced in Aspergillus oryzaeaccording to WO 02/095014) or 3% of total protein weight Aspergillusfumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzaeaccording to Example 22 of WO 02/095014) of cellulase protein loading isused as the source of the cellulolytic activity.

The polypeptides having cellulolytic enhancing activity have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 2.

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by proteins havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least0.1-fold, more at least 0.2-fold, more preferably at least 0.3-fold,more preferably at least 0.4-fold, more preferably at least 0.5-fold,more preferably at least 1-fold, more preferably at least 3-fold, morepreferably at least 4-fold, more preferably at least 5-fold, morepreferably at least 10-fold, more preferably at least 20-fold, even morepreferably at least 30-fold, most preferably at least 50-fold, and evenmost preferably at least 100-fold.

Cellulolytic activity: The term “cellulolytic activity” is definedherein as a biological activity which hydrolyzes a cellulosic material.Cellulolytic protein may hydrolyze or hydrolyzes carboxymethyl cellulose(CMC), thereby decreasing the viscosity of the incubation mixture. Theresulting reduction in viscosity may be determined by a vibrationviscosimeter (e.g., MIVI 3000 from Sofraser, France). Determination ofcellulase activity, measured in terms of Cellulase Viscosity Unit(CEVU), quantifies the amount of catalytic activity present in a sampleby measuring the ability of the sample to reduce the viscosity of asolution of carboxymethyl cellulose (CMC). The assay is performed at thetemperature and pH suitable for the cellulolytic protein and substrate.For CELLUCLAST™ (Novozymes NS. Bagsvaerd, Denmark) the assay is carriedout at 40° C. in 0.1 M phosphate pH 9.0 buffer for 30 minutes with CMCas substrate (33.3 g/L carboxymethyl cellulose Hercules 7 LFD) and anenzyme concentration of approximately 3.3-4.2 CEVU/ml. The CEVU activityis calculated relative to a declared enzyme standard, such as CELLUZYME™Standard 17-1194 (obtained from Novozymes Bagsvaerd, Denmark).

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by a cellulolytic mixture under the following conditions: 1-10mg of cellulolytic protein/g of cellulose in PCS for 5-7 day at 54° C.compared to a control hydrolysis without addition of cellulolyticprotein.

Endoglucanase: The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Cellobiohydrolase: The term “cellobiohydrolase” is defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain. For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem, 47: 273-279 and by van Tilbeurgh et al., 1982.FEBS Letters 149: 152-156: van Tilbeurgh and Claeyssens, 1985, FEBSLetters 187: 283-288. In the present invention, the Lever et al. methodwas employed to assess hydrolysis of cellulose in corn stover, while themethod of van Tilbeurgh at was used to determine the cellobiohydrolaseactivity on a fluorescent disaccharide derivative.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al. 2002, J. Basic Microbiol. 42: 55-66, exceptdifferent conditions were employed as described herein. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% TWEEN® 20.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” is defined herein as a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. Presently. Henrissat lists theGH61 Family as unclassified indicating that properties such asmechanism, catalytic nucleophile/base, catalytic proton donors, and 3-Dstructure are not known for polypeptides belonging to this family.

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant ishemi-cellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue The cellulosic material can beany type of biomass including, but not limited to, wood resources,municipal solid waste, wastepaper, crops, and crop residues (see, forexample, Wiselogel et al. 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 105-118, Taylor 8, Francis, Washington D.C.: Wyman,1994. Bioresource Technology 50: 3-16 Lynd, 1990, Applied Biochemistryand Biotechnology 24125: 695-719: Mosier et al., 1999, Recent Progressin Bioconversion of Lignocellulosics, in Advances in BiochemicalEngineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40. Springer-Verlag, New York). It is understood herein that thecellulose may be in the form of lignocellulose, a plant cell wallmaterial containing lignin, cellulose, and hemicellulose in a mixedmatrix. In a preferred aspect, the cellulosic material islignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotherpreferred aspect, the cellulosic material is corn fiber, in anotheraspect, the cellulosic material is corn cob. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material iswheat straw. In another aspect, the cellulosic material is switch grass.In another aspect, the cellulosic material is miscanthus. In anotheraspect, the cellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect the cellulosic material is pretreated.

Pre-treated corn stover: The term “PCS” or “Pre-treated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute acid.

Isolated polypeptide: The term “Isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e. that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide in its final form following translation and anypost-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. In apreferred aspect, the mature polypeptide is amino acids 16 to 310 of SEQID NO: 2 based on the SignalP program (Nielsen et al., 1997, ProteinEngineering 10:1-6) that predicts amino acids 1 to 15 of SEQ ID NO: 2are a signal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having cellulolytic enhancing activity. In apreferred aspect, the mature polypeptide coding sequence is nucleotides46 to 1250 of SEQ ID NO: 1 based on the SignalP program that predictsnucleotides 1 to 45 of SEQ ID NO: 1 encode a signal peptide.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al. 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein having an E value (or expectancy score) of less than0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols. S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Myceliophthora thermophila polypeptide having cellulolyticenhancing activity of SEQ ID NO: 2, or the mature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide of SEQ IDNO: 2; or a homologous sequence thereof; wherein the fragment hascellulolytic enhancing activity. In a preferred aspect, a fragmentcontains at least 250 amino acid residues, more preferably at least 265amino acid residues, and most preferably at least 280 amino acidresidues of the mature polypeptide of SEQ ID NO: 2 or a homologoussequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1;or a homologous sequence thereof; wherein the subsequence encodes apolypeptide fragment having cellulolytic enhancing activity. In apreferred aspect, a subsequence contains at least 750 nucleotides, morepreferably at least 795 nucleotides, and most preferably at least 840nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 ora homologous sequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention, Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2; or a homologous sequence thereof: as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be a substitution, a deletion and/or an insertion of one or more(several) amino acids as well as replacements of one or more (several)amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having cellulolytic enhancing activity produced byan organism expressing a modified polynucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1; or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the polynucleotide sequence disclosed inSEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellulolytic Enhancing Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence having a degree ofidentity to the mature polypeptide of SEQ ID NO: 2 of preferably atleast 60%, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have cellulolytic enhancingactivity (hereinafter “homologous polypeptides”). In a preferred aspect,the homologous polypeptides have an amino acid sequence that differs byten amino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having cellulolytic enhancing activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 16 to 310 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 16 to 310 of SEQ ID NO: 2. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 16 to 310 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 16 to 310 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under preferably very low stringencyconditions, more preferably low stringency conditions, more preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1,(iii) a subsequence of (i) or (ii), or (ivy a full-length complementarystrand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T,Maniatis, 1989, Molecular Cloning. A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.). A subsequence of the mature polypeptide codingsequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides orpreferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment having cellulolyticenhancing activity, in a preferred aspect, the complementary strand isthe full-length complementary strand of the mature polypeptide codingsequence of SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof;may be used to design nucleic acid probes to identify and clone DNAencoding polypeptides having cellulolytic enhancing activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes that are preferably at least 600nucleotides, more preferably at least 700 nucleotides, or mostpreferably at least 800 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having cellulolytic enhancing activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1;or a subsequence thereof; the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1;the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1; its full-length complementary strand; or a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1, in another preferred aspect, thenucleic acid probe is nucleotides 46 to 1250 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencethat encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pSMai193 which is contained in E. coliNRRL B-50086, wherein the polynucleotide sequence thereof encodes apolypeptide having cellulolytic enhancing activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pSMai193 which is contained in E. coli NRRLB-50086.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SOS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SOS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 of preferably at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide. See polynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NO: 2; or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly. Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,cellulolytic enhancing activity) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mot Biol. 224: 899-904: Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides that arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl, Aced. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837: U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Noret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, is 10, preferably9, more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide having cellulolytic enhancing activity of the presentinvention may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having cellulolytic enhancing activity, or a Gram negativebacterial polypeptide such as an E. coli. Pseudomonas, Salmonella,Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter,Neisseria, or Ureaplasma polypeptide having cellulolytic enhancingactivity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermaphilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avemitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enhancing activity.

A polypeptide having cellulolytic enhancing activity of the presentinvention may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having cellulolyticenhancing activity; or more preferably a filamentous fungal polypeptidesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Phanerochaete, Piromyces,Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide having cellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviform is polypeptide having cellulolytic enhancingactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium mops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia austrateinsis, Thielavia fimeti, Thielaviamicrospore, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptidehaving cellulolytic enhancing activity.

In another preferred aspect, the polypeptide is a Myceliophthorahinnulea, Myceliophthora lutea, Myceliophthora thermophila, orMyceliophthora vellerea polypeptide having cellulolytic enhancingactivity.

In a more preferred aspect, the polypeptide is a Myceliophthorathermophila polypeptide having cellulolytic enhancing activity. In amost preferred aspect, the polypeptide is a Myceliophthora thermophilaCBS 20235 polypeptide having cellulolytic enhancing activity, e.g., thepolypeptide comprising the mature polypeptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having cellulolytic enhancing activity from the fusionprotein. Examples of cleavage sites include, but are not limited to, aKex2 site that encodes the dipeptide Lys-Arg (Martin at al. 2003, J.Ind. Microbiol. Biotechnol. 3: 568-76: Svetina et al., 2000, J.Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ.Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503;and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al. 1986, Biochem. 25: 505-512): aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter at 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gin (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having cellulolytic enhancing activity of the presentinvention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pSMai193which is contained in E. coli NRRL B-50086. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 46 to 1250 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pSMai193 which is contained in E. coli NRRLB-50086. The present invention also encompasses nucleotide sequencesthat encode polypeptides comprising or consisting of the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differfrom SEQ ID NO: 1 or the mature polypeptide coding sequence thereof byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ IDNO: 2 that have cellulolytic enhancing activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2, respectively.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Myceliophthora, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 ofpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99% identity, which encode apolypeptide having cellulolytic enhancing activity.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labeling (see, e.g. de Vos et al.,1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii); or allelicvariants and subsequences thereof (Sambrook et al., 1989, supra), asdefined herein. In a preferred aspect, the complementary strand is thefull-length complementary strand of the mature polypeptide codingsequence of SEQ ID NO: 1.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, or (iii) a full-length complementary strand of (i) or (ii); and(b) isolating the hybridizing polynucleotide, which encodes apolypeptide having cellulolytic enhancing activity. In a preferredaspect, the complementary strand is the full-length complementary strandof the mature polypeptide coding sequence of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotidessequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94: and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei esparto proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase. Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase. Aspergillus nidulans acetamidase. Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I. Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I.Trichoderma reesei endoglucanase II. Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II. Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5″ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase. Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase. Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM, and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase. Rhizomucor miehei aspartic proteinase.Humicola insolens cellulase. Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al. 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 15 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 45 ofSEQ ID NO: 1.

The control sequence may also be a propeptide coding sequence that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propeptide is generallyinactive and can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression, in creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3. LEU2, LYS2, MET3, TRP1, and URA3 Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991. Gene 98: 61-67: Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175: WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium, Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Hyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformisBacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell, in another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E. coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g. Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacterial, 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbial. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g. Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ, Microbial, 65: 3800-3804) or by conjugation (see, e.g. Clewell,1981, Microbiol. Rev. 45: 409-436). However, any method known in the artfor introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge. UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M. and Davenport, R. R., ads, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookweilense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens.Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium imps, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983. Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Myceliophthora. In amore preferred aspect, the cell is Myceliophthora thermophila. In a mostpreferred aspect, the cell is Myceliophthora thermophila CBS 202.75. Inanother most preferred aspect, the cell is Myceliophthora thermophileCBS 117.65.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptide thatcomprises or consists of the mature polypeptide of SEQ ID NO: 2; and (b)Recovering the Polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides,

Plant

The present invention also relates to plants, e.g. a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having cellulolytic enhancing activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidapsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988. Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al. 1992, Plant Mot Biol. 18: 675-689; Zhang etal., 1991. Plant Cell 3: 1155-1165), Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994. Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998. Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248; 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293: Potrykus, 1990,Bio/Technology 8: 535: Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281: Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162: Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving cellulolytic enhancing activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA, sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of cellulolytic enhancingactivity by fermentation of a cell that produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibitingcellulolytic enhancing activity to the fermentation broth before,during, or after the fermentation has been completed, recovering theproduct of interest from the fermentation broth, and optionallysubjecting the recovered product to further purification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of cellulolytic enhancingactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce the cellulolyticenhancing activity substantially, and recovering the product from theculture broth. Alternatively, the combined pH and temperature treatmentmay be performed on an enzyme preparation recovered from the culturebroth. The combined pH and temperature treatment may optionally be usedin combination with a treatment with an cellulolytic enhancinginhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the cellulolytic enhancing activity. Complete removal ofcellulolytic enhancing activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thecellulolytic enhancing-deficient cells may also be used to expressheterologous proteins of pharmaceutical interest such as hormones,growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Methods of Inhibiting Expression of a Polypeptide

The present invention also relates to methods of inhibiting theexpression of a polypeptide in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of a polynucleotide of thepresent invention. In a preferred aspect, the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in acell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencingtherapeutics. In one aspect, the invention provides methods toselectively degrade RNA using the dsRNAis of the present invention. Theprocess may be practiced in vitro, ex vivo or in vivo. In one aspect,the dsRNA molecules can be used to generate a loss-of-function mutationin a cell, an organ or an animal. Methods for making and using dsRNAmolecules to selectively degrade RNA are well Known in the art, see, forexample, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No.6,515,109; and U.S. Pat. No. 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulose, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidogiutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans. Aspergillusniger, or Aspergillus oryzae, Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum.Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa or Trichoderma, preferably Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma wide.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art,

Processing of Cellulosic Material

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with a cellulolytic enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention in a preferred aspect, the method further comprises recoveringthe degraded or converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with a cellulolytic enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention; (b) fermenting the saccharified cellulosic material of step(a) with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with a cellulolytic enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention and the presence of the polypeptide having cellulolyticenhancing activity increases the degradation of the cellulosic materialcompared to the absence of the polypeptide having cellulolytic enhancingactivity. In a preferred aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another preferred aspect,the method further comprises recovering the fermentation product fromthe fermentation.

The composition comprising the polypeptide having cellulolytic enhancingactivity can be in the form of a crude fermentation broth with orwithout the cells removed or in the form of a semi-purified or purifiedenzyme preparation or the composition can comprise a host cell of thepresent invention as a source of the polypeptide having cellulolyticenhancing activity in a fermentation process with the biomass.

The methods of the present invention can be used to saccharify acellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., chemicals and fuels. Theproduction of a desired fermentation product from cellulosic materialtypically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); SHCF (separate hydrolysis andco-fermentation), HHCF (hybrid hydrolysis and fermentation), and directmicrobial conversion (DMC). SHF uses separate process steps to firstenzymatically hydrolyze lignocellulose to fermentable sugars, e.g.,glucose, cellobiose, cellobiose, and pentose sugars, and then fermentthe fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis oflignocellulose and the fermentation of sugars to ethanol are combined inone step (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212). SSCF involves thecofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999,Enzymes, energy and the environment; A strategic perspective on the U.S.Department of Energy's research and development activities forbioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separatehydrolysis separate step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures. i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, lignocellulose hydrolysis, and fermentation) in oneor more steps where the same organism is used to produce the enzymes forconversion of the lignocellulose to fermentable sugars and to convertthe fermentable sugars into a final product (Lynd, L. R., Weimer, P. J.van Zyl, W. H., and Pretorius, I. S., 2002, Microbial celluloseutilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.Reviews 66: 506-577), it is understood herein that any method known inthe art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza. Flavio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn. A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol, 7: 346-352), an attrition reactor (Ryu. S. K., and Lee,J. M. 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y. Davydkin, V. Y., Protas, O. V., 1996, Enhancementof enzymatic cellulose hydrolysis using a novel type of bioreactor withintensive stirring induced by electromagnetic field, Appl. Biochem.Biotechnol. 56: 141-153). Additional reactor types include: Fluidizedbed, upflow blanket, immobilized, and extruder type reactors forhydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt the plant cell wallcomponents. The cellulosic material can also be subjected topre-soaking, wetting, or conditioning prior to pretreatment usingmethods known in the art. Conventional pretreatments include, but arenot limited to, steam pretreatment (with or without explosion), diluteacid pretreatment, hot water pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment, Additional pretreatmentsinclude ultrasound, electroporation, microwave, supercritical CO₂,supercritical H₂O, and ammonia percolation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with hydrolysis, such as simultaneously with treatment ofthe cellulosic material with one or more cellulolytic enzymes, or otherenzyme activities, to release fermentable sugars, such as glucose and/ormaltose. In most cases the pretreatment step itself results in someconversion of biomass to fermentable sugars (even in absence ofenzymes).

Steam Pretreatment.

In steam pretreatment, the cellulosic material is heated to disrupt theplant cell wall components, including lignin, hemicellulose, andcellulose to make the cellulose and other fractions, e.g.,hemicellulase, accessible to enzymes. The lignocellulose material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatthe cellulosic material is generally only moist during the pretreatment.The steam pretreatment is often combined with an explosive discharge ofthe material after the pretreatment, which is known as steam explosion,that is, rapid flashing to atmospheric pressure and turbulent flow ofthe material to increase the accessible surface area by fragmentation(Duff and Murray, 1996, Bioresource Technology 855: 1-33: Galbe andZacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. PatentApplication No. 20020164730). During steam pretreatment, hemicelluloseacetyl groups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem, Biotechnol. 129-132: 496-508: Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment:

The term “chemical treatment” refers to any chemical pretreatment thatpromotes the separation and/or release of cellulose, hemicellulose,and/or lignin. Examples of suitable chemical pretreatment processesinclude, for example, dilute acid pretreatment, lime pretreatment, wetoxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation(APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulosic material is mixed withdilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999. Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005. Bioresource Technol 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol 117: 1-17; Varga et al., 2004, Biotechnol Bioeng. 88:567-574; Martin et al., 2006. J. Chem. Technol. Biotechnol 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem, Biotechnol 98: 23-35: Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231: Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121:1133-1141: Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi at 2005, Appl. Biochem.Biotechnol. 121:219-230). Sulphuric acid is usually added as a catalyst.In organosolv pretreatment, the majority of the hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al. 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulosic material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment. The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment:

The term “physical pretreatment” refers to any pretreatment thatpromotes the separation and/or release of cellulose, hemicellulose,and/or lignin from cellulosic material. For example, physicalpretreatment can involve irradiation (e.g., microwave irradiation),steaming/steam explosion, hydrothermolysis, and combinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulosic material canbe pretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof to promote the separation and/or release of cellulose,hemicellulose and/or lignin.

Biological Pretreatment:

The term “biological pretreatment” refers to any biological pretreatmentthat promotes the separation and/or release of cellulose, hemicellulose,and/or lignin from the cellulosic material. Biological pretreatmenttechniques can involve applying lignin-solubilizing microorganisms (see,for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis. Washington, D.C., 179-212; Ghosh and Singh, 1993,Physicochemical and biological treatments for enzymatic/microbialconversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333;McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, inEnzymatic Conversion of Biomass for Fuels Production, Himmel, M. E.,Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566,American Chemical Society, Washington, D.C., chapter 15: Gong, C. S.,Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology. Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production.Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990.Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the pretreatedcellulosic material is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,xylose, xylulose, arabinose, maltose, mannose, galactose, or solubleoligosaccharides. The hydrolysis is performed enzymatically by acellulolytic enzyme composition comprising a polypeptide havingcellulolytic enhancing activity of the present invention, which canfurther comprise one or more hemicellulolytic enzymes. The enzymes ofthe compositions can also be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt. %, and most preferably about 20 toabout 30 wt.

In addition to a polypeptide having cellulolytic enhancing activity ofthe present invention, the cellulolytic enzyme components of thecomposition are preferably enzymes having endoglucanase,cellobiohydrolase, and beta-glucosidase activities. In a preferredaspect, the cellulolytic enzyme composition comprises one or more(several) cellulolytic enzymes selected from the group consisting of acellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase. Inanother preferred aspect, the cellulolytic enzyme preparation issupplemented with one or more additional enzyme activities selected fromthe group consisting of hemicellulases, esterases (e.g., lipases,phospholipases, and/or cutinases), proteases, laccases, peroxidases, ormixtures thereof. In the methods of the present invention, theadditional enzyme(s) can be added prior to or during fermentation,including during or after propagation of the fermentingmicroorganism(s).

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more amino acids that are deleted, inserted and/or substituted,i.e. a recombinantly produced enzyme that is a mutant and/or a fragmentof a native amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained recombinantly, such as by site-directedmutagenesis or shuffling.

The enzymes used in the present invention can be in any form suitablefor use in the methods described herein, such as a crude fermentationbroth with or without cells or substantially pure polypeptides. Theenzyme(s) can be a dry powder or granulate, a non-dusting granulate, aliquid, a stabilized liquid, or a protected enzyme(s). Granulates can beproduced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661.452,and can optionally be coated by process known in the art. Liquid enzymepreparations can, for instance, be stabilized by adding stabilizers suchas a sugar, a sugar alcohol or another polyol, and/or lactic acid oranother organic acid according to established process. Protected enzymescan be prepared according to the process disclosed in EP 238,216

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosicsubstrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme(s) tocellulosic material is about 0.5 to about 50 mg, preferably at about 0.5to about 40 mg, more preferably at about 0.5 to about 25 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 0.5 to about 10 mg, andmost preferably at about 2.5 to about 10 mg per g of cellulosicmaterial.

In another preferred aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulosic material is about 0.01 toabout 50 mg, preferably at about 0.5 to about 40 mg, more preferably atabout 0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg,more preferably at about 0.75 to about 15 mg, even more preferably atabout 0.5 to about 10 mg, and most preferably at about 2.5 to about 10mg per g of cellulosic material.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulosic material is about0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, morepreferably about 0.01 to about 30 mg, more preferably about 0.01 toabout 20 mg, more preferably about 0.01 to about 10 mg, more preferablyabout 001 to about 5 mg, more preferably at about 0.025 to about 1.5 mg,more preferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulolytic enzyme(s) isabout 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g,more preferably at about 0.15 to about 0.75 g, more preferably at about0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, evenmore preferably at about 0.1 to about 0.5 g, and most preferably atabout 0.05 to about 0.2 g per g of cellulolytic enzyme(s).

Fermentation.

The fermentable sugars obtained from the pretreated and hydrolyzedcellulosic material can be fermented by one or more fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous. Such methods include, but are not limited to, separatehydrolysis and fermentation (SHF); simultaneous saccharification andfermentation (SSF); simultaneous saccharification and cofermentation(SSCF); hybrid hydrolysis and fermentation (HHF); SHCF (separatehydrolysis and co-fermentation), HHCF (hybrid hydrolysis andfermentation), and direct microbial conversion (DMC).

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention, include cellulosicmaterials, such as wood or plant residues or low molecular sugars DP1-3obtained from processed cellulosic material that can be metabolized bythe fermenting microorganism, and which can be supplied by directaddition to the fermentation medium.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment. i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbial. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast, Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicatis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Klyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae, in another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P. 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTARTT™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, App. Biochem, Biotechnol, 39-40: 135-147: Hoet al., 1998. Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ, Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303: Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomanas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded lignocellulose or hydrolysate and the fermentation isperformed for about 12 to about 96 hours, such as typically 24-60 hours.In a preferred aspect, the temperature is preferably between about 20°C. to about 60° C., more preferably about 25° C. to about 50° C., andmost preferably about 32° C. to about 50° C., in particular about 32° C.or 50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some, e.g. bacterial fermentingorganisms have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenoreat al., Improving ethanol production and viability of Saccharomycescerevisiae by a vitamin feeding strategy during fed-batch process,Springer-Verlag (2002), which is hereby incorporated by reference.Examples of minerals include minerals and mineral salts that can supplynutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid): a ketone (e.g.,acetone); an amino acid (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); and a gas (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)). The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J. Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid, inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid, inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example. Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy. Vol. 13 (1-2), pp. 83-114,1997. Anaerobic digestion of biomass for methane production: A review.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Cellulolytic Enzyme Compositions

In the methods of the present invention, the cellulolytic enzymecomposition may comprise any protein involved in the processing of acellulose-containing material to glucose, or hemicellulose to xylose,mannose, galactose, and arabinose, their polymers, or products derivedfrom them as described below. In one aspect, the cellulolytic enzymecomposition comprises one or more enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the cellulolytic enzyme compositionfurther comprises one or more additional enzyme activities to improvethe degradation of the cellulose-containing material. Preferredadditional enzymes are hemicellulases, esterases (e.g., lipases,phospholipases, and/or cutinases), proteases, laccases, peroxidases, ormixtures thereof.

The cellulolytic enzyme composition may be a monocomponent preparation,e.g., an endoglucanase, a multicomponent preparation, e.g.,endoglucanase(s), cellobiohydrolase(s), and beta-glucosidase(s), or acombination of multicomponent and monocomponent protein preparations.The cellulolytic proteins may have activity, i.e. hydrolyze thecellulose-containing material, either in the acid, neutral, or alkalinepH-range.

As mentioned above, the cellulolytic proteins used in the presentinvention may be monocomponent preparations, i.e., a componentessentially free of other cellulolytic components. The single componentmay be a recombinant component, i.e., produced by cloning of a DNAsequence encoding the single component and subsequent cell transformedwith the DNA sequence and expressed in a host (see, for example. WO91/17243 and WO 91/17244). The host cell may be a heterologous host(enzyme is foreign to host) or the host may also be a wild-type host(enzyme is native to host). Monocomponent cellulolytic proteins may alsobe prepared by purifying such a protein from a fermentation broth.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

A polypeptide having cellulolytic enzyme activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterocaccus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enzymeactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campytobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus.Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenzyme activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enzyme activity.

The polypeptide having cellulolytic enzyme activity may also be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having cellulolytic enzyme activity; or more preferably afilamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula. Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having cellulolyticenzyme activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis. Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenzyme activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium Mops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspore, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma Trichoderma longibrachiatum, Trichoderma reesei, Trichodermaviride, or Trichaphaea saccata polypeptide having cellulolytic enzymeactivity.

Chemically modified or protein engineered mutants of cellulolyticproteins may also be used.

One or more components of the cellulolytic enzyme composition may be arecombinant component, produced by cloning of a DNA sequence encodingthe single component and subsequent cell transformed with the DNAsequence and expressed in a host (see, for example, WO 91/17243 and WO91/17244). The host is preferably a heterologous host (enzyme is foreignto host), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

Examples of commercial cellulolytic protein preparations suitable foruse in the present invention include, for example, CELLUCLAST™(available from Novozymes A/S) and NOVOZYM™ 188 (available fromNovozymes A/S). Other commercially available preparations comprisingcellulase that may be used include CELLUZYME™, CEREFLO™ and ULTRAFLO™(Novozymes A/S), LAMINEX™ and SPEZYME™ CP (Genencor int.), ROHAMENT™7069 W (Röhm GmbH), and FIBREZYME® LDI, FiBREZYME® LBR, or VISCOSTAR®150L (Dyadic international, inc., Jupiter, Fla., USA). The cellulaseenzymes are added in amounts effective from about 0.001% to about 5.0%wt. of solids, more preferably from about 0.025% to about 4.0% wt. ofsolids, and most preferably from about 0.005% to about 2.0% wt. ofsolids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the methods of thepresent invention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo,et al., 1988, Gene 63:11-22; GENBANK™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al, 1988, App. Environ. Microbiol.64: 555-563; GENBANK™ accession no, AB003694); Trichoderma reeseiendoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591;GENBANK™ accession no. Y11113): and Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al.1990, Nucleic Acids Research 18: 5884); Aspergillus kawachiiendoglucanase (Sakamoto of al., 1995, Current Genetics 27: 435-439);Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381);Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no.AB003107): Melanocarpus albomyces endoglucanase (GENBANK™ accession no.MAL515703); Neurospora crassa endoglucanase (GENBANK™ accession no,XM_(—)324477): Humicola insolens endoglucanase V (SEQ ID NO: 17);Myceliophthora thermophila CBS117.65 endoglucanase (SEQ ID NO: 19);basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 21); basidiomyceteCBS 494.95 endoglucanase (SEQ ID NO: 23); Thielavia terrestris NRRL 8126CEL6B endoglucanase (SEQ ID NO: 25); Thielavia terrestris NRRL 8126CEL6C endoglucanase (SEQ ID NO: 27); Thielavia terrestris NRRL 8126CEL7C endoglucanase (SEQ ID NO: 29); Thielavia terrestris NRRL 8126CEL7E endoglucanase (SEQ ID NO: 31); Thielavia terrestris NRRL 8126CEL7F endoglucanase (SEQ ID NO: 33); Cladorrhinum foecundissimum ATCC62373 CEL7A endoglucanase (SEQ ID NO: 35); and Trichoderma reesei strainNo. VTT-D-88133 endoglucanase (SEQ ID NO: 37; GENBANK™ accession no.M15665). The endoglucanases of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27. SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37 described aboveare encoded by the mature polypeptide coding sequence of SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQID NO: 36, respectively.

Examples of cellobiohydrolases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseicellobiohydrolase I (SEQ ID NO: 39); Trichoderma reeseicellobiohydrolase II (SEQ ID NO: 41); Humicola insolenscellobiohydrolase I (SEQ ID NO: 43), Myceliophthora thermophilacellobiohydrolase II (SEQ ID NO: 45 and SEQ ID NO: 47), Thielaviaterrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 49), Chaetomiumthermophilum cellobiohydrolase I (SEQ ID NO: 51), and Chaetomiumthermophilum cellobiohydrolase II (SEQ ID NO: 53). Thecellobiohydrolases of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO:53 described above are encoded by the mature polypeptide coding sequenceof SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52, respectively.

Examples of beta-glucosidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus oryzaebeta-glucosidase (SEQ ID NO: 55): Aspergillus fumigatus beta-glucosidase(SEQ ID NO: 57); Penicillium brasilianum IBT 20888 beta-glucosidase (SEQID NO: 59); Aspergillus niger beta-glucosidase (SEQ ID NO: 61); andAspergillus aculeatus beta-glucosidase (SEQ ID NO: 63). Thebeta-glucosidases of SEQ ID NO: 55, SEQ ID NO: 57. SEQ ID NO: 59, SEQ IDNO: 61, and SEQ ID NO: 63 described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, and SEQ ID NO: 62, respectively.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein of SEQ ID NO: 65 or the Aspergillus oryzaebeta-glucosidase fusion protein of SEQ ID NO: 67. In another aspect, theAspergillus oryzae beta-glucosidase variant BG fusion protein is encodedby the polynucleotide of SEQ ID NO: 64 or the Aspergillus oryzaebeta-glucosidase fusion protein is encoded by the polynucleotide of SEQID NO: 66.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531.372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619. WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078. WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432. WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

The cellulolytic enzymes used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, Calif., 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and cellulolytic enzymeproduction are known in the art (see, e.g., Bailey, J. E., and Ollis, D.F., Biochemical Engineering Fundamentals. McGraw-Hill Book Company, NY,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic enzyme. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe cellulolytic enzyme to be expressed or isolated. The resultingcellulolytic enzymes produced by the methods described above may berecovered from the fermentation medium and purified by conventionalprocedures.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to anucleotide sequence encoding a signal peptide comprising or consistingof amino acids 1 to 15 of SEQ ID NO: 2, wherein the gene is foreign tothe nucleotide sequence.

In a preferred aspect, the nucleotide sequence comprises or consists ofnucleotides 1 to 45 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods of producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides that comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more (several) may be heterologous or native to the hostcell. Proteins further include naturally occurring allelic andengineered variations of the above mentioned proteins and hybridproteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strain

Myceliophthora thermophila CBS 202.75 was used as the source of a Family61 gene encoding a polypeptide having cellulolytic enhancing activity.

Media

BA medium was composed per liter of 10 g of corn steep liquor drymatter, 10 g of NH₄NO₃, 10 g of KH₂PO₄, 0.75 g of MgSO₄.7H₂O: 0.1 ml ofplutonic, and 0.5 g of CaCO₅. The pH was adjusted to 6.5 beforeautoclaving.

YEG medium was composed per liter of 20 g of dextrose and 5 g of yeastextract.

Minimal medium plates were composed per liter of 6 g of NaNO₃, 0.52 g ofKCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g ofNoble agar, 20 ml of 50% glucose, 2.5 ml of MgSO₄.7H₂O, and 20 ml of a0.02% biotin solution.

COVE trace metals solution was composed per liter of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

M410 medium was composed per liter of 50 g of maltose, 50 g of glucose,2 g of MgSO₄.7H₂O, 2 g of KH₂PO₄, 4 g of anhydrous citric acid, 8 g ofyeast extract, 2 g of urea, 0.5 g of CaCl₂, and 0.5 ml of AMG tracemetals solution.

AMG trace metals was composed per liter of 14.3 g of ZnSO₄.7H₂O, 2.5 gof CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.7H₂O, and 3 g of citric acid.

Example 1 Identification of Family 61 Peptides

SDS-PAGE Analysis.

A commercial product was diluted 1:10 with water. Twenty μl wasseparated on a CRITERION™ 8-16% Tris-HCl SDS-PAGE gel according to themanufacturer's suggested conditions (Bio-Rad Laboratories, Hercules,Calif., USA). PRECISION PLUS PROTEIN™ standards (Bio-Rad Laboratories,Hercules, Calif., USA) were used as molecular weight markers. The gelwas stained with BIO-SAFE™ Coomassie Stain (Bio-Rad Laboratories,Hercules, Calif., USA), and visible bands were excised with a razorblade for protein identification analysis.

In-Gel Digestion of Polypeptides for Peptide Sequencing.

A MultiPROBE® II Liquid Handling Robot (PerkinElmer Life and AnalyticalSciences, Boston, Mass., USA) was used to perform the in-gel digestions.Gel bands containing protein were reduced with 50 μl of 10 mMdithiothreitol (DTT) in 100 mM ammonium bicarbonate pH 8.0 for 30minutes. Following reduction, the gel piece was alkylated with 50 μl of55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 8.0 for 20minutes. The dried gel piece was allowed to swell in 25 μl of a trypsindigestion solution (6 ng/μl sequencing grade trypsin (Promega, Madison,Wis., USA) in 50 mM ammonium bicarbonate pH 8 for 30 minutes at roomtemperature, followed by an 8 hour digestion at 40° C. Each of thereaction steps described above was followed by numerous washes andpre-washes with the appropriate solutions following the manufacturer'sstandard protocol. Fifty μl of acetonitrile was used to de-hydrate thegel piece between reactions and the gel piece was air dried betweensteps. Peptides were extracted twice with 1% formic acid/2% acetonitrilein HPLC grade water for 30 minutes. Peptide extraction solutions weretransferred to a 96 well skirted PCR type plate (ABGene, Rochester,N.Y., USA) that had been cooled to 10-15° C. and covered with a 96-wellplate lid (PerkinElmer Life and Analytical Sciences, Boston, Mass., USA)to prevent evaporation. Plates were further stored at 4° T. until massspectrometry analysis could be performed.

Protein Identification.

For de novo peptide sequencing by tandem mass spectrometry, a Q-TOFMICRO™ (Waters Micromass MS Technologies, Milford, Mass., USA), a hybridorthogonal quadrupole time-of-flight mass spectrometer was used forLC/MS/MS analysis. The Q-TOF MICRO™ is fully microprocessor controlledusing MASSLYNX™ software version 4.1 (Waters Micromass MS Technologies,Milford, Mass., USA). The Q-TOF MICRO™ was fitted with an ULTIMATEcapillary and nano-flow HPLC system, which was coupled with a FAMOS™micro autosampler and a SWITCHOS™ II column switching device(LCPackings/Dionex, Sunnyvale, Calif., USA) for concentrating anddesalting samples. Samples were loaded onto a guard column (300 μm ID×5cm. PEPMAP™ C18) fitted in the injection loop and washed with 0.1%formic acid in water at 40 μl per minute for 2 minutes using a SwitchosII pump. Peptides were separated on a 75 μm ID×15 cm. C18, 3 μm, 100 ÅPEPMAP™ (LC Packings, San Francisco, Calif., USA) nanoflow fusedcapillary column at a flow rate of 175 μl/minute from a split flow of175 μl/minute using a NAN-75 calibrator (Dionex, Sunnyvale, Calif.,USA). A step elution gradient of 5% to 80% acetonitrile in 0.1% formicacid was applied over a 45 minute interval. The column eluent wasmonitored at 215 nm and introduced into the Q-TOF MICRO™ through anelectrospray ion source fitted with the nanospray interface.

Data was acquired in survey scan mode and from a mass range of m/z 400to 1990 with switching criteria for MS to MS/MS to include an ionintensity of greater than 10.0 counts per second and charge states of+2, +3, and +4. Analysis spectra of up to 4 co-eluting species with ascan time of 1.9 seconds and inter-scan time of 0.1 seconds could beobtained. A cone voltage of 45 volts was typically used and thecollision energy was programmed to be varied according to the mass andcharge state of the eluting peptide and in the range of 10-60 volts. Theacquired spectra were combined, smoothed, and centered in an automatedfashion and a peak list generated. This peak list was searched againstselected databases using PROTEINLYNX™ Global Server 2.2.05 software(Waters Micromass MS Technologies, Milford, Mass., USA) and PEAKS Studioversion 4.5 (SP1) (Bioinformatic Solutions Inc., Waterloo, Ontario,Canada) Results from the PROTEINLYNX™ and PEAKS Studio searches wereevaluated and un-identified proteins were analyzed further by evaluatingthe MS/MS spectra of each ion of interest and de novo sequence wasdetermined by identifying the y and b ion series and matching massdifferences to the appropriate amino acid.

Peptide sequences were obtained from several multiply charged ions forthe in-gel digested approximately 24 kDa polypeptide gel band. A doublycharged tryptic peptide ion of 871.56 m/z sequence was determined to be[Leu]-Pro-Ala-Ser-Asn-Ser-Pro-Val-Thr-Asp-Val-Thr-Ser-Asn-Ala-[Leu]-Arg(SEQ ID NO: 3). A doubly charged tryptic peptide ion of 615.84 m/zsequence was determined to beVal-Asp-Asn-Ala-Ala-Thr-Ala-Ser-Pro-Ser-Gly-[Leu]-Lys (SEQ ID NO: 4). Adoubly charged tryptic peptide ion of 715.44 m/z sequence was determinedto be [Leu]-Pro-Ala-Asp-[Leu]-Pro-Ser-Gly-Asp-Tyr-[Leu]-[Leu]-Arg (SEQID NO: 5). A doubly charged tryptic peptide ion of 988.58 m/z sequencewas determined to be Gly-Pro-[Leu]-[Gln]-Val-Tyr-[Leu]-Ala-Lys (SEQ IDNO: 6). A double charged tryptic peptide ion of 1272.65 m/z sequence wasdetermined to be Val-Ser-Val-Asn-Gly-[Gln]-Asp-[Gln]-Gly-[Gln]-[Leu]-Lys(SEQ ID NO: 7). [Leu] above may be He or Leu and [Gln] above may be Glnor Lys because they could not be distinguished due to equivalent masses.

Example 2 Preparation of Myceliophthora thermophila CBS 117.65 cDNA Pool

Myceliophthora thermophila CBS117.65 was cultivated in 200 ml of BAmedium at 30° C. for five days at 200 rpm. Mycelia from the shake flaskculture were harvested by filtering the contents through a funnel linedwith MIRACLOTH™ (CalBiochem, San Diego, Calif., USA). The mycelia werethen sandwiched between two MIRACLOTH™ pieces and blotted dry withabsorbent paper towels. The mycelial mass was then transferred toplastic centrifuge tubes and frozen in liquid nitrogen. Frozen myceliawere stored in a −80° C. freezer until use.

The extraction of total RNA was performed with guanidinium thiocyanatefollowed by ultracentrifugation through a 5.7 M CsCl cushion, andisolation of poly(A)+ RNA was carried out by oligo(dT)-celluloseaffinity chromatography, using the procedures described in WO 94/14953.

Double-stranded cDNA was synthesized from 5 μg of poly(A)+ RNA by theRNase H method (Gubler and Hoffman, 1983, Gene 25: 263-269, Sambrook etal., 1989, Molecular cloning: A laboratory manual, Cold Spring Harborlab., Cold Spring Harbor, N.Y., USA). The poly(A)+ RNA (5 μg in 5 μl ofDEPC (0.1% diethylpyrocarbonate)-treated water) was heated at 70° C. for8 minutes in a pre-siliconized, RNase-free EPPENDORF® tube, quenched onice, and combined in a final volume of 50 μl with reverse transcriptasebuffer composed of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol (DTT) (Bethesda Research Laboratories, Bethesda, Md.,USA), 1 mM of dATP, dGTP and dTTP, and 0.5 mM 5-methyl-dCTP (GEHealthcare, Piscataway, N.J., USA), 40 units of human placentalribonuclease inhibitor (RNasin; Promega, Madison, Wis., USA), 1.45 μg ofoligo(dT)18-Not I primer (GE Healthcare, Piscataway, N.J., USA), and1000 units of SuperScript II RNase H reverse transcriptase (BethesdaResearch Laboratories, Bethesda, Md., USA). First-strand cDNA wassynthesized by incubating the reaction mixture at 45° C. for 1 hour.After synthesis, the mRNA:cDNA hybrid mixture was gel filtrated througha MICROSPIN™ S-400 HR spin column (GE Healthcare, Piscataway, N.J., USA)according to the manufacturers instructions.

After gel filtration, the hybrids were diluted in 250 μl of secondstrand buffer (20 mM Tris-HCl, pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂SO₄, 0.16 mM NAD) containing 200 μM of each dNTP, 60 units of E.coli DNA polymerase I (GE Healthcare, Piscataway, N.J., USA), 5.25 unitsof RNase H (Promega, Madison, Wis., USA), and 15 units of E. coli DNAligase (Boehringer Mannheim, Manheim, Germany). Second strand cDNAsynthesis was performed by incubating the reaction tube at 16° C. for 2hours and an additional 15 minutes at 25° C. The reaction was stopped byaddition of EDTA to a final concentration of 20 mM followed by phenoland chloroform extractions.

The double-stranded cDNA was precipitated at −20° C. for 12 hours byaddition of 2 volumes of 96% ethanol and 0.2 volume of 10 M ammoniumacetate, recovered by centrifugation at 13.000×g, washed in 70% ethanol,dried, and resuspended in 30 μl of Mung bean nuclease buffer (30 mMsodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35 mM OTT, 2%glycerol) containing 25 units of Mung bean nuclease (GE Healthcare,Piscataway, N.J., USA). The single-stranded hair-pin DNA was clipped byincubating the reaction at 30° C. for 30 minutes, followed by additionof 70 μl of 10 mM Tris-HCl-1 mM EDTA pH 7.5, phenol extraction, andprecipitation with 2 volumes of 96% ethanol and 0.1 volume of 3 M sodiumacetate pH 5.2 on ice for 30 minutes.

The double-stranded cDNAs were recovered by centrifugation at 13,000×gand blunt-ended in 30 μl of T4 DNA polymerase buffer (20 mMTris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate,1 mM DTT) containing 0.5 mM of each dNTP and 5 units of T4 DNApolymerase (New England Biolabs, Ipswich, Mass., USA) by incubating thereaction mixture at 16° C. for 1 hour. The reaction was stopped byaddition of EDTA to a final concentration of 20 mM, followed by phenoland chloroform extractions, and precipitation for 12 hours at −20° C. byadding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH5.2. After the fill-in reaction the cDNAs were recovered bycentrifugation at 13,000×g, washed in 70% ethanol, and dried.

Example 3 Myceliophthora thermophila CBS 202.75 and Myceliophthorathermophila CBS 117.65 Genomic DNA Extraction

Myceliophthora thermophila CBS 202.75 and Myceliophthora thermophila CBS117.65 strains were grown in 100 ml of YEG medium in a baffled shakeflask at 45° C. and 200 rpm for 2 days, Mycelia were harvested byfiltration using MIRACLOTH® (Calbiochem. La Jolla. Calif., USA), washedtwice in deionized water, and frozen under liquid nitrogen. Frozenmycelia were ground, by mortar and pestle, to a fine powder, and totalDNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia,Calif., USA).

Example 4 Molecular Screening of a Family 61 Gene from Myceliophthorathermophila

Degenerate primers were designed, as shown below, based upon peptidesequences obtained through tandem mass spectrometry as described inExample 1.

Primer 061564 (CI61B sense):

5′-TCTCGGTCAACGGCCAGGAYCARGGNCA-3′ (SEQ ID NO: 8)Primer 061565 (CI61B anti):

5′-GCGAGGCGGTGGCGGCRTTRTCNACYTT-3′ (SEQ D NO: 9)

Fifty picomoles each of CI61B sense and CI61B anti primers were used ina PCR reaction composed of 100 ng of Myceliophthora thermophila CBS202.75 genomic DNA, or Myceliophthora thermophila CBS 117.65 cDNA poolDNA, 1× ADVANTAGE® GC-Melt LA Buffer (Clontech Laboratories, Inc.,Mountain View, Calif., USA), 0.4 mM each of dATP, dTTP, dGTP, and dCTP,and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix (ClontechLaboratories, Inc., Mountain View, Calif., USA) in a final volume of 25μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed for 1cycle at 94° C. for 1 minutes; and 30 cycles each at 94° C. for 30seconds, 56.5° C. for 30 seconds, and 72° C. for 30 seconds, followed bya final extension of 5 minutes at 72° C.

The reaction products were fractionated by 1% agarose gelelectrophoresis in 40 mM Tris base-20 mM sodium acetate-1 mM disodiumEDTA TAE) buffer and bands of greater than 300 bp were excised, purifiedusing a MINELUATE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.,USA) according to the manufacturers instructions, and subcloned using aTOPO® TA Kit (Invitrogen, Carlsbad, Calif., USA). Plasmid DNA wasextracted from a number of E. coli transformants and sequenced. Sequenceanalysis of the E. coli clones showed that the sequences contained acoding region of a Family 61 gh61i gene.

Example 5 Isolation of a Full-Length Family 61 Gene (gh61i) fromMyceliophthora thermophila CBS 202.75

A full-length Family 61 gb61i gene from Myceliophthora thermophila CBS202.15 was isolated using a GENOMEWALKER™ Universal Kit (ClontechLaboratories, Inc., Mountain View, Calif., USA) according to themanufacturer's instructions. Briefly, total genomic DNA fromMyceliophthora thermophila CBS 202.75 was digested separately with fourdifferent restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) thatleave blunt ends. Each batch of digested genomic DNA was then ligatedseparately to the GENOMEWALKER™ Adaptor (Clontech Laboratories. Inc.,Mountain View, Calif., USA) to create four libraries. These librarieswere then employed as templates in PCR reactions using gene-specificprimers for the Myceliophthora thermophila Family 61 gh61i gene. Theprimers shown below were designed based on the partial Family 61 gh61igene sequences obtained in Example 4.

Upstream Region Primers:

(SEQ ID NO: 10) MtGH61I-R1:  5′-AGGCGGCGATCGGGTTGTCCGGGTCGTT-3′(SEQ ID NO: 11) MtGH61I-R2: 5′-GTTGCAGGCCATGTTGGCATCGTTGACG-3′Downstream Region Primers:

(SEQ ID NO: 12) MtGH61I-F1: 5′-GTCGAGCAACTCCCCGATCCAGAACGTCAAC-3′(SEQ ID NO: 13) MtGH61I-F2: 5′-GACCCGGACAACCCGATCGCCGCCTCCCACAA-3′

Two primary PCR amplifications were performed, one to isolate theupstream region and the other the downstream region of theMyceliophthora thermophila gh61i gene. Each PCR amplification (25 μl)was composed of 1 μl (approximately 6 ng) of each library as template,0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 μmol of Adaptor Primer 1(Clontech Laboratories, Inc., Mountain View, Calif., USA), 10 μmol ofprimer MtGH61I-R1 or primer MtGH61I-F1, 1× ADVANTAGE® GC-Melt LA Buffer,and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix. Theamplifications were performed using an EPPENDORF® MASTERCYCLER® 5333programmed for pre-denaturing at 94° C. for 1 minute; 7 cycles each at adenaturing temperature of 94° C. for 30 seconds; annealing andelongation at 72° C. for 5 minutes; and 32 cycles each at a denaturingtemperature of 94° C. for 30 seconds; annealing and elongation 67° C.for 5 minutes, followed by a final extension of 7 minutes at 67° C.

The secondary amplifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmolof Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif.,USA), 10 pmol of nested primer MtGH61I-R2 or MtGH61I-F2, 1× ADVANTAGESGC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC Genomic PolymeraseMix in a final volume of 25 μl. The amplifications were performed usingan EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C.for 1 minute; 5 cycles each at a denaturing temperature of 94° C. for 30seconds; annealing and elongation at 72° C. for 5 minutes; and 20 cycleseach at a denaturing temperature of 94° C. for 30 seconds; annealing andelongation at 67° C. for 5 minutes, followed by a final extension of 7minutes at 67° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TAE buffer where a 2 kb PCR product (upstream region) from the Stu Ilibrary and a 1.2 kb PCR fragment (downstream region) from the Eco RVlibrary were excised from the gel, purified using a MINELUTE® GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturers instructions. The PCR products were sequenced directly orsubcloned using a TOPO® TA Kit and then sequenced.

Example 6 Characterization of the Myceliophthora thermophila GenomicSequence Encoding a Family GH61I Polypeptide Having CellulolyticEnhancing Activity

DNA sequencing of the PCR fragments was performed with a Perkin-ElmerApplied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) usingdye-terminator chemistry (Giesecke at al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. Nucleotide sequence datawere scrutinized for quality and all sequences were compared to eachother with assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash., USA).

A gene model for the Myceliophthora thermophila GH61I polypeptide havingcellulolytic enhancing activity was constructed based on similarity ofthe encoded protein to homologous glycoside hydrolase Family 61 proteinsfrom Thielavia terrestris (accession numbers GENESEQP:ADM97933.GENESEQP:AEB90517), Chaetomium globosum (UNIPROT:Q2HGH1, UNIPROT:Q2GW98)and Neurospora crassa (UNIPROT:Q7S439). To verify the sequenceinformation obtained for the Myceliophthora thermophila gh61i gene, afurther PCR reaction was carried out using a pair of gene specificprimers (shown below), which encompass the complete gene.

Primer MtGH61I-F5:

(SEQ ID NO: 14) 5′-ACTGGATTTACCATGAAGCCTTTTAGCCTCGTCGCC-3′Primer MtGH61 l-R3:

(SEQ ID NO: 15) 5′-TCACCTCTAGTTAATTAACTAGGGGAGGCACTGGCTGT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2 (WO 2004/099228).

The PCR consisted of fifty picomoles of forward and reverse primers in aPCR reaction composed of 100 ng of Myceliophthora thermophila CBS 202.75genomic DNA, Pfx Amplification Buffer (Invitrogen, Carlsbad. Calif.,USA), OA mM each of dATP, dTTP, dGTP, and dCTP, 1 mM MgCl₂ and 2.5 unitsof Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA) in a finalvolume of 50 μl. The amplification were performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 98° C. for 3 minutes; and30 cycles each at 98° C. for 30 seconds, 60° C. for 30 seconds, and 72°C. for 1.5 minutes, followed by a final extension of 15 minutes at 72°C. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TAE buffer and purified using a MINELUTE® Gel Extraction Kitaccording to the manufacturer's instructions. In order to clone the PCRfragments into pCR®2.1-TOPO® vector (Invitrogen, Carlsbad, Calif., USA),addition of 3′ A-overhangs was performed using Tag DNA polymerase (NewEngland Biolabs, Ipswich, Mass., USA).

A 1293 bp Myceliophthora thermophila gh61i gene fragment was cloned intopCR®2.1-TOPO® vector using a TOPO® TA Cloning Kit to generate pSMai193(FIG. 2).

The Myceliophthora thermophila gh61i insert was confirmed by DNAsequencing. E. coli pSMai193 was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., USA, on Dec. 5, 2007, and assigned accessionnumber B-50086.

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Myceliophthora thermophila GH61I polypeptidehaving cellulolytic enhancing activity are shown in FIG. 1. The genomicpolynucleotide encodes a polypeptide of 310 amino acids, interrupted by2 introns of 91 and 229 bp. The % G+C content of the full-length codingsequence and the mature coding sequence are 62.2% and 68.0%,respectively. Using the SignalP software program (Nielsen et al., 1997,Protein Engineering 10:1-6), a signal peptide of 15 residues waspredicted. The predicted mature protein contains 295 amino acids with amolecular mass of 30.2 kDa.

Analysis of the deduced amino acid sequence of the GH61I polypeptidehaving cellulolytic enhancing activity with the Interproscan program(Mulder et al., 2007, Nucleic Acids Res. 35: D224-D228) showed that theGH61I polypeptide contained the sequence signature of the fungalcellulose-binding domain (InterPro accession IPRO00254). This sequencesignature was found from approximately residues 277 to 309 of the maturepolypeptide (SMART accession number SM00236).

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Ma Blot, 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Myceliophthora thermophila GH61I mature polypeptideshared 83.2% identity (excluding gaps) to the deduced amino acidsequences of a Family 61 glycoside hydrolase protein from Neurosporacrassa (Uniprot accession number Q7S439).

Example 7 Construction of an Aspergillus oryzae Expression VectorContaining Myceliophthora thermophila CBS 202.75 Genomic SequenceEncoding a Family GH16I Polypeptide Having Cellulolytic EnhancingActivity

The same 1293 bp Myceliophthora thermophila gh61i PCR fragment generatedin Example 6 was cloned into Nco I and Pac I digested pAILo2 (WO2004/099228) using an Infusion Cloning Kit (BD Biosciences, Palo Alto,Calif., USA) resulting in pSMai189 (FIG. 3) in which transcription ofthe Myceliophthora thermophila gh61i gene was under the control of ahybrid of promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase (NA2-tpipromoter). The ligation reaction (50 μl) was composed of 1× InFusionBuffer (BD Biosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences,Palo Alto, Calif., USA), 1 μl of Infusion enzyme (diluted 1:10) (BDBiosciences, Palo Alto, Calif., USA), 100 ng of pAILo2 digested with NcoI and Pac I, and 50 ng of the Myceliophthora thermophila gh61i purifiedPCR product. The reaction was incubated at room temperature for 30minutes. One μl of the reaction was used to transform E. coli XL10SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, Calif., USA).An E. coli transformant containing pSMai189 was detected by restrictiondigestion and plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGENInc., Valencia, Calif., USA). The Myceliophthora thermophila gh61iinsert in pSMai189 was confirmed by DNA sequencing.

Example 13 Expression of the Myceliophthora thermophila Family 61Glycosyl Hydrolase Genes (gh61i) in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422, Three μg of pSMai189 were transformed into the Aspergillusoryzae JaL355 protoplasts.

Twenty transformants were isolated to individual Minimal medium platesfrom the transformation experiment.

Confluent Minimal Medium plates of each of the transformants were washedwith 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml ofM410 medium in 125 ml glass shake flasks and incubated at 34° C., 250rpm. After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® 8-16% Tris-HCl SDS-PAGE gels with a CRITERION®Cell (Bio-Rad Laboratories, Hercules, Calif., USA), according to themanufacturer's instructions. The resulting gels were stained withBIO-SAFE™ Coomassie Stain (Bio-Rad Laboratories, Hercules, Calif., USA).SDS-PAGE profiles of the cultures showed that the majority of thetransformants had the expected band size of 30 kDa.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria. Ill., 61604, USA, and given the followingaccession number:

Deposit Accession Number Date of Deposit E. coli pSMai193 NRRL B-50086Dec. 5, 2007

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulotylic enhancing activity,selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 60%identity to the mature polypeptide of SEQ ID NO; 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, or (iii) afull-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 60% identity to the mature polypeptide codingsequence of SEQ ID NO: 1; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

[2] The polypeptide of paragraph 1, comprising an amino acid sequencehaving at least 60% identity to the mature polypeptide of SEQ ID NO: 2.

[3] The polypeptide of paragraph 2, comprising an amino acid sequencehaving at least 65% identity to the mature polypeptide of SEQ ID NO: 2.

[4] The polypeptide of paragraph 3, comprising an amino acid sequencehaving at least 70% identity to the mature polypeptide of SEQ ID NO: 2.

[5] The polypeptide of paragraph 4, comprising an amino acid sequencehaving at least 75% identity to the mature polypeptide of SEQ ID NO: 2.

[6] The polypeptide of paragraph 5, comprising an amino acid sequencehaving at least 80% identity to the mature polypeptide of SEQ ID NO: 2.

[7] The polypeptide of paragraph 6, comprising an amino acid sequencehaving at least 85% identity to the mature polypeptide of SEQ ID NO: 2.

[8] The polypeptide of paragraph 7, comprising an amino acid sequencehaving at least 90% identity to the mature polypeptide of SEQ ID NO: 2.

[9] The polypeptide of paragraph 8, comprising an amino acid sequencehaving at least 95% identity to the mature polypeptide of SEQ ID NO: 2.

[10] The polypeptide of paragraph 1, comprising or consisting of theamino acid sequence of SEQ ID NO: 2; or a fragment thereof havingcellulolytic enhancing activity.

[11] The polypeptide of paragraph 10, comprising or consisting of theamino acid sequence of SEQ ID NO: 2.

[12] The polypeptide of paragraph 10, comprising or consisting of themature polypeptide of SEQ ID NO: 2.

[13] The polypeptide of paragraph 1, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[14] The polypeptide of paragraph 13, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[15] The polypeptide of paragraph 14, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[16] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 60%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[17] The polypeptide of paragraph 16, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 65%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[18] The polypeptide of paragraph 17, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 70%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 75%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[20] The polypeptide of paragraph 19, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 80%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[21] The polypeptide of paragraph 20, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 85%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[22] The polypeptide of paragraph 21, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 90%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[23] The polypeptide of paragraph 22, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 95%identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

[24] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1: or a subsequence thereof encoding a fragment havingcellulolytic enhancing activity.

[25] The polypeptide of paragraph 24, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1.

[26] The polypeptide of paragraph 24, which is encoded by apolynucleotide comprising or consisting of the mature polypeptide codingsequence of SEQ ID NO: 1.

[27] The polypeptide of paragraph 1, wherein the polypeptide is avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[28] The polypeptide of paragraph 1, which is encoded by thepolynucleotide contained in plasmid pSMai193 which is contained in E.coli NRRL B-50086.

[29] The polypeptide of any of paragraphs 1-28, wherein the maturepolypeptide is amino acids 16 to 310 of SEQ IQ NO: 2.

[30] The polypeptide of any of paragraphs 1-29, wherein the maturepolypeptide coding sequence is nucleotides 46 to 1250 of SEQ ID NO: 1.

[31] An isolated polynucleotide comprising a nucleotide sequence thatencodes the polypeptide of any of paragraphs 1-30.

[32] The isolated polynucleotide of paragraph 31, comprising at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,in which the mutant nucleotide sequence encodes the mature polypeptideof SEQ ID NO: 2.

[33] A nucleic acid construct comprising the polynucleotide of paragraph31 or 32 operably linked to one or more (several) control sequences thatdirect the production of the polypeptide in an expression host.

[34] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 33.

[35] A recombinant host cell comprising the nucleic acid construct ofparagraph 33.

[36] A method of producing the polypeptide of any of paragraphs 1-30,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[37] A method of producing the polypeptide of any of paragraphs 1-30,comprising: (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[38] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a nucleotide sequence encoding the polypeptide ofany of paragraphs 1-30, which results in the mutant producing less ofthe polypeptide than the parent cell.

[39] A mutant cell produced by the method of paragraph 38.

[40] The mutant cell of paragraph 39, further comprising a gene encodinga native or heterologous protein.

[41] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 40 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[42] The isolated polynucleotide of paragraph 31 or 32, obtained by (a)hybridizing a population of DNA under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii); and (b) isolating the hybridizing polynucleotide, whichencodes a polypeptide having cellulolytic enhancing activity.

[43] The isolated polynucleotide of paragraph 42, wherein the maturepolypeptide coding sequence is nucleotides 46 to 1250 of SEQ ID NO: 1.

[44] A method of producing a polynucleotide comprising a mutantnucleotide sequence encoding a polypeptide having cellulolytic enhancingactivity, comprising: (a) introducing at least one mutation into themature polypeptide coding sequence of SEQ ID NO: 1, wherein the mutantnucleotide sequence encodes a polypeptide comprising or consisting ofthe mature polypeptide of SEQ ID NO: 2; and (b) recovering thepolynucleotide comprising the mutant nucleotide sequence.

[45] A mutant polynucleotide produced by the method of paragraph 44.

[46] A method of producing a polypeptide, comprising: (a) cultivating acell comprising the mutant polynucleotide of paragraph 45 encoding thepolypeptide under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[47] A method of producing the polypeptide of any of paragraphs 1-30,comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

[48] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-30.

[49] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 31 or 32, whereinoptionally the dsRNA is a siRNA or a miRNA molecule.

[50] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph49, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[51] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell a double-stranded RNA (dsRNA)molecule, wherein the dsRNA comprises a subsequence of thepolynucleotide of paragraph 31 or 32.

[52] The method of paragraph 51, wherein the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[53] A nucleic acid construct comprising a gene encoding a proteinoperably linked to a nucleotide sequence encoding a signal peptidecomprising or consisting of amino acids 1 to 15 of SEQ ID NO: 2, whereinthe gene is foreign to the nucleotide sequence.

[54] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 53.

[55] A recombinant host cell comprising the nucleic acid construct ofparagraph 53.

[56] A method of producing a protein, comprising: (a) cultivating therecombinant host cell of paragraph 55 under conditions conducive forproduction of the protein; and (b) recovering the protein.

[57] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide having cellulolyticenhancing activity of any of paragraphs 1-30, wherein the presence ofthe polypeptide having cellulolytic enhancing activity increases thedegradation of cellulosic material compared to the absence of thepolypeptide having cellulolytic enhancing activity.

[58] The method of paragraph 57, wherein the cellulosic material ispretreated.

[59] The method of paragraph 57 or 58, wherein the cellulolytic enzymecomposition comprises one or more cellulolytic enzymes are selected fromthe group consisting of a cellulase, endoglucanase, cellobiohydrolase,and beta-glucosidase.

[60] The method of any of paragraphs 57-59, further comprising treatingthe cellulosic material with one or more enzymes selected from the groupconsisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[61] The method of any of paragraphs 57-60, further comprisingrecovering the degraded cellulosic material.

[62] The method of paragraph 61, wherein the degraded cellulosicmaterial is a sugar.

[63] The method of paragraph 62, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[64] A method for producing a fermentation product, comprising;

(a) saccharifying a cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide having cellulolyticenhancing activity of any of paragraphs 1-20, wherein the presence ofthe polypeptide having cellulolytic enhancing activity increases thedegradation of cellulosic material compared to the absence of thepolypeptide having cellulolytic enhancing activity;

(b) fermenting the saccharified cellulosic material of step (a) with oneor more fermenting microorganisms to produce the fermentation product;and

(c) recovering the fermentation product from the fermentation.

[65] The method of paragraph 64, wherein the cellulosic material ispretreated.

[66] The method of paragraph 64 or 65, wherein the cellulolytic enzymecomposition comprises one or more cellulolytic enzymes selected from thegroup consisting of a cellulase, endoglucanase, cellobiohydrolase, andbeta-glucosidase.

[67] The method of any of paragraphs 64-66, further comprising treatingthe cellulosic material with one or more enzymes selected from the groupconsisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[68] The method of any of paragraphs 64-67, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[69] The method of any of paragraphs 64-68, wherein the fermentationproduct is an alcohol, organic acid, ketone, amino acid, or gas.

[70] A method of fermenting a cellulosic material, comprising;fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with acellulolytic enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of any of paragraphs 1-30 and thepresence of the polypeptide having cellulolytic enhancing activityincreases the degradation of the cellulosic material compared to theabsence of the polypeptide having cellulolytic enhancing activity.

[71] The method of paragraph 70, wherein the fermenting of thecellulosic material produces a fermentation product.

[72] The method of paragraph 71, further comprising recovering thefermentation product from the fermentation.

[73] The method of any of paragraphs 70-72, wherein the cellulosicmaterial is pretreated before saccharification.

[74] The method of any of paragraphs 70-73, wherein the cellulolyticenzyme composition comprises one or more cellulolytic enzymes selectedfrom the group consisting of a cellulose, endoglucanase,cellobiohydrolase, and beta-glucosidase.

[75] The method of any of paragraphs 70-74, wherein the cellulolyticenzyme composition further comprises one or more enzymes selected fromthe group consisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[76] The method of any of paragraphs 70-75, wherein the fermentationproduct is an alcohol, organic acid, Ketone, amino acid, or gas.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. An isolated polypeptide having cellulolyticenhancing activity, wherein the polypeptide is selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to amino acids 16-310 of SEQ IDNO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) nucleotides 46-1250of SEQ ID NO: 1, (ii) a cDNA sequence contained in nucleotides 46-1250of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or(ii); and (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 99% sequence identity to nucleotides46-1250 of SEQ ID NO:
 1. 2. The polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:
 2. 3. The polypeptide of claim 1,which is encoded by the polynucleotide contained in plasmid pSMai193,wherein a representative sample of E. coli bacteria comprising pSMai187has been deposited under NRRL Accession number B-50086.
 4. A nucleicacid construct comprising a nucleotide sequence that encodes thepolypeptide of claim 1 operably linked to one or more heterologouscontrol sequences that direct the production of the polypeptide in anexpression host.
 5. A recombinant host cell comprising the nucleic acidconstruct of claim
 4. 6. A method of producing a polypeptide, saidmethod comprising: (a) cultivating an isolated cell which produces thepolypeptide of claim 1 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.
 7. A method ofproducing a polypeptide, said method comprising: (a) cultivating therecombinant host cell of claim 5 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 8. Amethod of producing a polypeptide, said method comprising: (a)cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the polypeptide of claim 1 under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.
 9. A transgenic plant, plant part or plant cell transformedwith a polynucleotide encoding the polypeptide of claim 1, wherein thepolypeptide comprises a signal peptide directing the polypeptide intothe secretory pathway.
 10. A nucleic acid construct comprising a proteincoding sequence operably linked to a nucleotide sequence encoding asignal peptide comprising amino acids 1 to 15 of SEQ ID NO: 2, whereinthe protein coding sequence is foreign to the nucleotide sequence thatencodes the signal peptide.
 11. A recombinant host cell comprising thenucleic acid construct of claim
 10. 12. A method of producing a protein,said method comprising: (a) cultivating the recombinant host cell ofclaim 11 under conditions conducive for production of the protein; and(b) recovering the protein.
 13. A method for degrading or converting acellulosic material, said method comprising treating the cellulosicmaterial with a cellulolytic enzyme composition in the presence of thepolypeptide of claim 1, wherein the presence of the polypeptideincreases the degradation of cellulosic material compared to the absenceof the polypeptide.
 14. The method of claim 13, said method furthercomprising recovering the degraded cellulosic material.
 15. A method forproducing a fermentation product, said method comprising: (a)saccharifying a cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide of claim 1, wherein thepresence of the polypeptide increases the degradation of cellulosicmaterial compared to the absence of the polypeptide; (b) fermenting thesaccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.
 16. A method offermenting a cellulosic material, said method comprising: fermenting thecellulosic material with one or more fermenting microorganisms, whereinthe cellulosic material is saccharified with a cellulolytic enzymecomposition in the presence of a polypeptide of claim 1 and the presenceof the polypeptide increases the degradation of the cellulosic materialcompared to the absence of the polypeptide.
 17. The polypeptide of claim1, comprising an amino acid sequence having at least 95% sequenceidentity to amino acids 16-310 of SEQ ID NO:
 2. 18. The polypeptide ofclaim 1, comprising an amino acid sequence having at least 97% sequenceidentity to amino acids 16-310 of SEQ ID NO:
 2. 19. The polypeptide ofclaim 1, comprising an amino acid sequence having at least 98% sequenceidentity to amino acids 16-310 of SEQ ID NO:
 2. 20. The polypeptide ofclaim 1, comprising an amino acid sequence having at least 99% sequenceidentity to amino acids 16-310 of SEQ ID NO:
 2. 21. The polypeptide ofclaim 1, comprising amino acids 16-310 of SEQ ID NO:
 2. 22. A nucleicacid construct comprising an isolated polynucleotide comprising anucleotide sequence encoding the polypeptide of claim 21 operably linkedto one or more heterologous control sequences that direct production ofthe polypeptide in an expression host.
 23. A recombinant host cellcomprising the nucleic acid construct of claim
 22. 24. A method ofproducing a polypeptide, said method comprising: (a) cultivating therecombinant host cell of claim 23 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. 25.The polypeptide of claim 1, encoded by a polynucleotide that hybridizesunder at least high stringency conditions with the full-lengthcomplementary strand of (i) nucleotides 46-1250 of SEQ ID NO: 1, or (ii)a cDNA sequence contained in nucleotides 46-1250 of SEQ ID NO:
 1. 26.The polypeptide of claim 1, encoded by a polynucleotide that hybridizesunder at least very high stringency conditions with the full-lengthcomplementary strand of (i) nucleotides 46-1250 of SEQ ID NO: 1, or (ii)a cDNA sequence contained in nucleotides 46-1250 of SEQ ID NO:
 1. 27.The polypeptide of claim 1, encoded by a polynucleotide that comprises anucleotide sequence having at least 99% sequence identity to nucleotides46-1250 of SEQ ID NO:
 1. 28. The polypeptide of claim 1, encoded by apolynucleotide that comprises nucleotides 46-1250 of SEQ ID NO: 1.